The incidence of endometriosis is not known accurately but is estimated at about 10% in women. Endometriosis is a significantly under-diagnosed and under-treated, disease. Diagnosis is usually made by visualization of endometriosis lesions by invasive surgery through laparoscopy or laparotomy with histological confirmation (Pasoto et al. 2008; Baidi et al, 2008).
Endometriosis is a benign gynaecological proliferative and inflammatory disease in which endometrial or endometrial-like tissue, which normally forms the uterine lining shed in menstruation, is present outside of its normal place in the uterus. Endometriosis growths occur most commonly in the pelvis, around the outside of the uterus, on the ovaries, the fallopian tubes, but are also found on the bowel, the bladder, the intestines, the vagina and the rectum. Endometriosis tissue may also occur in the muscle layer of the wall of the uterus (adenomyosis). Rarely, endometriosis tissue may grow in other areas including the skin, the eyes, the spine, the lungs and the brain. Although not a cancer, endometriosis has certain characteristics in common with benign tumours including progressive growth, invasive growth, oestrogen-dependent growth, recurrence and a tendency to metastasize (Van Gorp et al. 2004; Flores et al., 2007).
The normal endometrium lining of the uterus proliferates and thickens during the luteal phase of the menstrual cycle in response to rising circulatory levels of progesterone and estradiol. When the progesterone and estradiol levels fall at the end of the cycle (in the absence of fertilization) the endometrium breaks down and bleeds and is shed in menstruation, before re-growing when progesterone and estradiol levels rise again in the next cycle. Ectopic, endometriosis tissue is similarly influenced by the hormones of the menstrual cycle and grows and breaks down with bleeding. However, as this tissue is located outside of the uterus, the bleeding is internal with no way of leaving the body leading to inflammation, pain, and the formation of scar tissue (adhesions).
Symptoms of endometriosis may include pain, infertility, dysmenorrhoea and fatigue (D'Hooghe and Hummelshoj, 2006; D'hooghe of et al., 2006). The severity of the symptoms varies with the position of the endometriosis growths and the severity and stage of the disease. The severity may also increase with the total life-time number of menstrual cycles experienced by the patient. For this reason early diagnosis and early treatment to limit cycles is important. Endometriosis was thought to be rare in adolescents but is now more commonly diagnosed (Templeman, 2009). Under diagnosis or late diagnosis is common because endometriosis symptoms are non-specific and because many patients are asymptomatic. Endometriosis often remains undetected for a number of years.
The etiology of the disease is not known but a number of theories have been put forward. One theory involves anatomical or biochemical aberrations of uterine function; for example the implantation of menstrual tissue on pelvic organs following backwash of this tissue into the pelvis possibly due to vaginal blockage of outflow. Another theory involves small defects of embryogenesis in the foetus where endometrial-like tissue is developmental misplaced. Other theories involve the travel of menstrual tissue through veins or lymphatic, vessels to other sites or the differentiation of blood cells originating in the bone marrow into endometrial tissue at various sites (Bulun, 2009; Signorile et al., 2009).
Treatment of the disease is by removal of the endometriosis tissue surgically, often in the same operation in which diagnosis is made, or by the use of drugs including androgens (such as Danazol), GnRH agonists (such as Leuprolide, Burserelin, Goserelin or Nafarelin), progestagens (such as Gestrinone or Medroxyprogesterone) and oral contraceptives.
Diagnosis of endometriosis is currently made by inspection of the pelvis by laparoscopy. If growths are identified simultaneous treatment may foe performed (Kennedy et al., 2005). However, many patients with mild disease remain undiagnosed. There is a need for non-invasive or minimally invasive methods for endometriosis diagnosis to facilitate early treatment and to reduce the number of unnecessary laparoscopes performed (D'Hooghe et al, 2006; Kennedy, 2006).
Many methods for the diagnosis of endometriosis have been investigated but visualization of the lesions by invasive surgery through laparoscopy or laparotomy remains the method of choice with a clinical sensitivity, of approximately 98% and a clinical specificity of approximately 79% (Baldi et al, 2008; de Almeida Filho et al, 2008).
Biomarkers investigated as diagnostic tools for endometriosis include circulating DNA, the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor α, angiogenic factors, such as IL-8 and vascular endothelial growth factor and the tumour markers CA-125 and CA 19-9 (Seeber, 2009). Despite research to identify circulatory biomarkers for endometriosis, there is currently no clinically applicable blood test available for the detection of endometriosis (Zachariah et at, 2009; Seeber et al 2009).
As single biomarkers have proved ineffective to date as diagnostic fools in endometriosis, combinations of biomarkers that might together prove to foe diagnostic of the disease have been investigated. Serum concentrations of C-reactive protein and high sensitivity C-reactive protein are reported to foe of little use as a diagnostic tool for endometriosis (Lermann et al, 2009). Measurements of a combination of CCR1 mRNA in peripheral blood leukocytes and monocyte chemotactic protein-1 (MCP-1) and CA125 protein in serum has been investigated as a possible diagnostic test for endometriosis. The expression of CCR1 mRNA in peripheral blood leukocytes was measured by quantitative real-time polymerase chain reaction. MCP-1 and CA125 levels in serum were determined by ELISA and ECLIA. The method was reported to have a sensitivity of 92% and a specificity of 82% (Agic et al. 2008).
Mass spectrometry has been used to screen for proteins expressed differently in serum from patients with endometriosis versus normal controls, in one recent study three protein markers identified were measured to produce a sensitivity of 92%, and specificity of 75% (Zhang et al. 2009). In another study six proteins were measured to produce a test that identified approximately two thirds of endometriosis patients (Seeber, 2009).
Other minimally invasive methods investigated for the diagnosis of endometriosis include global gene analysis of the eutopic endometrium at late secretory phase (Sherwin et al, 2008) and patient response to preoperative hormonal therapy in terms of relief of chronic pelvic pain (Jenkins et al, 2008), but neither of these approaches has proved effective.
Elevated levels of circulating nucleosomes have been defected in the blood of some endometriosis patients outdo not discriminate between healthy and diseased subjects (Holdenrieder et al, 2001), Elevated levels of circulating DNA have also been detected in the blood, of some endometriosis patients and this has been investigated as a diagnostic measurement for the detection of endometriosis and found to have a clinical sensitivity of 70% and a clinical specificity of 87% (Zachariah et al, 2009, Zachariah et. al, 2008)
Two particular problems for blood assays of cell death products, including DNA (Zachariah et al, 2009) and nucleosomes (Holdenrieder et al, 2001), for use as diagnostic tools for the detection of endometriosis are that;                (i) they have poor clinical sensitivity. Whilst elevated levels are found in some patients suffering with endometriosis, other patients with endometriosis do not have elevated levels. This leads to the misdiagnosis of many patients suffering with endometriosis as being disease free.        (ii) they have poor clinical specificity. Whilst elevated levels are found in some patients suffering with endometriosis, elevated levels also occur in many other clinical conditions including malignant and benign tumours, autoimmune conditions, inflammatory conditions, and trauma. This means that not all positive results are due to endometriosis and the tests misdiagnose many patients with other conditions wrongly as suffering with endometriosis.        
Similarly problems occur for the use of markers of inflammation, such as C-reactive protein, high sensitivity C-reactive protein, fibrinogen amyloid A and inflammatory cytokines such as IL-1, IL-6, IL-8 and tumour necrosis factor, soluble intercellular adhesion molecule or white blood cell count (Lermann et al, 2008). CA-125 has also been measured as a potential diagnostic marker for endometriosis and, although more often used as a tumour marker, is known to be associated with inflammation. These markers have been found to be raised in many, but not all, patients with endometriosis and may be raised in other conditions leading to low clinical sensitivity and specificity.
Due to these problems some workers in the field have tried to produce blood tests for endometriosis with improved clinical sensitivity and specificity following one or both of two broad strategies;                (i) workers have measured combinations of a number of markers in the expectation that measurement of two or several or many different markers in the same patient would provide improved discrimination between patients with and without endometriosis. Recent examples of this approach include the measurement of CCR1 mRNA in peripheral blood leukocytes and monocyte chemotactic protein-1 (MCP-1) and CA125 protein in serum (Agic et al, 2008), a combination of three protein biomarkers (Zhang et al, 2009) and a combination of six protein biomarkers (Seeber 2009).        (ii) workers have measured markers in samples taken at different phases within the menstrual cycle in the expectation that the discrimination provided by the marker level between patients with and without endometriosis may be improved by making the measurements in samples taken during a particular phase of the menstrual cycle, rather than samples taken at other phases or in untimed samples. A recent example of this approach involved the measurement of IL-1 receptor agonist in serum and peritoneal fluid in endometriosis patients during the proliferative and the secretory phases of the menstrual cycle, IL-1 receptor agonist was found to be lower in the peritoneal fluid of endometriosis patients than control patients but no such difference was found in serum. Moreover, no difference was found in the levels of IL-1 receptor agonist in samples taken during the secretory or proliferative phase of the menstrual cycle in either serum or peritoneal fluid samples (Zhang et al, 2007). This indicates that no advantage is gained by measurement of IL-1 receptor agonist in timed samples taken during a particular phase of the menstrual cycle.        
Similarly, serum IL-12 and IL-18 levels were measured during the follicular and luteal phases of the menstrual cycle. IL-12 was raised in advanced endometriosis over control subjects but no such difference was observed for IL-18. Neither IL-12 nor IL-18 levels varied between the follicular and luteal phases of the menstrual cycle, either in endometriosis patients or in control subjects (Fairbanks et al. 2009).                It has been reported that serum levels of CA-125, C-Reactive Protein, amyloid A, and anti-cardiolipin antibodies are on average elevated in endometriosis patients during Days 1-3 of the menstrual cycle (the first 3 days of menses). The levels of these markers were also measured during Days 8-10 of the menstrual cycle and were also elevated, but on average less elevated than during Days 1-3. Of these measurements serum CA-125 levels during Days 1-3 were reported to be the best predictor of advanced endometriosis although not for early disease. Clinical specificity was not determined as comparison was made with healthy patients and not tested, with patients suspected of having endometriosis but found to be disease free on laparoscopy (Abrao et al, 1997). This study was later extended to include the measurement of CA-15-3, CA-19-9, CEA, AFP and B2MG during Days 1-3 and 8-10. None of these additional markers were found to be discriminatory for endometriosis (Abrao et al, 1999) either during Days 1-3 or during Days 8-10.        (iii) Some workers have combined the previous two approaches and measured multiple markers for endometriosis at different phases of the menstrual cycle in the expectation that measurement of multiple markers in the same patient would provide improved discrimination between patients with and without endometriosis and that this discrimination may be further improved by making those measurements in timed samples taken at a particular phase of the menstrual cycle. A recent example includes the measurement of six serum cytokines as predictors of endometriosis. The levels of three of these cytokines were found to be raised in endometriosis, over levels found in healthy patients. The best discriminator was found to be IL-6 measurement with sensitivity of 71% and specificity of 66%, Discrimination was not improved by the inclusion of other cytokine measurements. Measurements were taken during the proliferative and secretory phases of the menstrual cycle. No difference was found in the serum cytokine levels of any of the six markers when measured at different phases of the menstrual cycle. This indicates that no advantage is gained by measurement of multiple cytokines over IL-6 alone and that discrimination is not improved by making the measurements in samples taken during a particular phase of the menstrual cycle (Othman et al, 2008).        
Another example of this approach involved the measurement of a plurality of biomarkers in samples taken morn patients in a determined phase of the menstrual cycle and the analysis of the concentrations found using a mathematical model to determine the presence or absence or degree of disease, in this approach the workers measured multiple biomarkers in samples taken during different phases (in particular the proliferative and secretory phases) of the menstrual cycle. The aim of this was to maximise disease discrimination by the inclusion of multiple biomarkers and measuring those biomarkers at the phase of the cycle where their individual and combined discrimination is maximised. The secretory phase was selected as the phase where the greatest discrimination was found and a computer programme was used to set multiple inter-dependent cutt-off points to maximise clinical sensitivity and specificity (WO 2008/049175).
None of these methods has been adopted in clinical practice and a non-invasive blood test for endometriosis remains a clear and unmet medical need (Zachariah et al, 2009; Seeber et al, 2000).